Whole-brain mapping of afferent and efferent connections of lateral hypothalamic orexinergic neurons in mice.

Orexin (Orx) is a vital peptide neurotransmitter essential for regulating feeding, sleep-wake cycles, and reward-seeking behavior. Orexinergic neurons are predominantly located in the lateral hypothalamus (LH). However, the precise neural connectivity of these neurons across the brain remains insufficiently characterized. In this study, we used cell-specific anterograde and retrograde viral tracers to map the afferent and efferent connections of LH orexinergic neurons. Our results revealed that LH orexinergic neurons received extensive afferent inputs from cortical regions, including the primary motor cortex (M1) and secondary motor cortex (M2), the basal ganglia, including the caudate putamen (CPu), the diencephalon, such as the zona incerta (ZI) and reticular nucleus (Rt), and brainstem nuclei, including the ventral tegmental area (VTA) and locus coeruleus (LC). Simultaneously, the orexinergic neurons projected primarily to the telencephalon, including the bed nucleus of the stria terminalis (BNST) and lateral septum (LS), the diencephalon, including the medial preoptic nucleus (MPA) and lateral preoptic area (LPO), and the brainstem, such as the periaqueductal gray (PAG) and superior colliculus (SC). Among these, BNST, LS, MPA, and PAG were the primary projection targets of LH orexinergic neurons, whereas ZI, VTA, M1, and LC serve as their major input sources. Additionally, significant bidirectional projections were observed between LH orexinergic neurons and regions such as ZI and VTA, suggesting their potential roles in signal integration. These findings provide a detailed framework for the neural connectivity of LH orexinergic neurons, highlighting their potential involvement in mediating complex brain functions and neuropsychiatric disorders.